Project 4: Therapeutic Gene Editing for Rett Syndrome

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PROJECT SUMMARY PROJECT 4 (Rett) Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the gene MECP2. The majority of RTT is caused by the same d eight reoccurring mutations in mutational hotspots of the 3rd and 4th exons. These four missense mutations and four nonsense mutations (R106W, R133C, T158M, R168X, R255X, R270X, R294X, and R306C) make up 70% of all Rett cases. RTT predominantly affects females, occurring at an incidence of 1-10,000 live female births and presents as a regression in milestones between the ages of 6-18 months. Hallmark characteristics of Rett include progressive loss of purposeful hand skills, speech and language regression, gait abnormalities, and stereotypic hand movements. Other features of the disorder include decelerated head growth, sleep disturbances, breathing abnormalities, and a high incidence of seizures. The life expectancy for patients with Rett is typically around 40-50 years of age. Landmark studies using conditional mouse models of Mecp2 to turn on the endogenous Mecp2 mouse gene post-symptomatically have convincingly demonstrated that neurological deficits associated with loss of Mecp2 is reversible to a significant degree. To date, clinical trials have focused on small molecules that modulate mechanisms downstream of MECP2. With more the 25+ such trials, limited success has been demonstrated with only modest behavior modifications and no approved FDA drugs for RTT. Gene and protein replacement strategies have been stymied with regards to controlled dosage affects that require precise titration of MECP2, as overexpression of Mecp2 in cells is toxic and are further confounded by the X-linked nature of the disease with cell to cell variation of MECP2 expression resulting from chromosome X-linked inactivation. For these reasons, base editing strategies that directly correct the endogenous genetic mutation and restore MECP2 to endogenous cellular levels are an extremely attractive therapeutic strategy for RTT. In this follower project, we aim to correct missense and nonsense mutations to enable rescue of disease progression in Rett patients. Specifically, we aim to: (1) Design, test and optimize Base Editing, Prime editing and Twin Editing strategies for 5 different mutations; (2) Test these strategies in mouse models containing humanized exons with engineered missense and nonsense mutations. (3) Perform pre-clinical IND enabling studies to assess safety and efficacy. We will work closely with the Gene Editing Core to develop the latest base editing and/or prime editing technologies in RTT model systems. We will iterate with the Gene Editing Core to ensure that our genome editing tools maximize on-target editing efficiencies, minimize undesirable gene editing byproducts and off-target editing events, and maximize compatibility with in vivo delivery methods of potential therapeutic relevance.